Copper chelating cyclic peptidomimetic inhibits Aβ fibrillogenesis

Abstract

Misfolding of the amyloid-β peptide (Aβ) and its subsequent aggregation into toxic oligomers is one of the leading causes of Alzheimer's disease (AD). As a therapeutic approach, cyclic peptides have been modified in many ways and developed as a potential class of amyloid aggregation inhibitors. Head-to-tail cyclic peptides with alternating D, L amino acids inhibit amyloid aggregation significantly. On the other hand, excess deposition of copper, iron, and zinc enhances amyloid aggregation. Dysregulation of these metal ions in the brain triggers aggregation by binding to the Aβ peptide. Therefore, specific metal chelators have been developed for disrupting the Aβ-metal complex, thereby reducing toxicity and restoring metal ion homeostasis. Herein, we report the development of a head-to-tail cyclic peptidomimetic with a copper chelating ligand attached. The designed peptidomimetic inhibits amyloid aggregation significantly in a two-fold molar ratio to the Aβ peptide, as confirmed by the thioflavin T (ThT) fluorescence assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), and Congo-red stained birefringence studies. The chelating ligand attached to the cyclic peptide binds efficiently to Cu²⁺ but weakly to Zn²⁺ and Fe²⁺, thereby exhibiting profound selectivity, probed using UV-visible spectroscopy, thioflavin T (ThT) fluorescence assay, tyrosine (TYR10) fluorescence assay, isothermal titration calorimetry (ITC) and transmission electron microscopy (TEM). The non-toxicity of the designed peptidomimetics and their ability to reduce aggregating Aβ-fragment induced cytotoxicity was confirmed by the MTT assay on the mouse neuronal cell line. Further, the molecular interaction between the peptidomimetics and the Aβ-fragment was confirmed by Förster resonance energy transfer (FRET) studies using fluorescently labeled analogs. Cytotoxicity and cell internalization were also confirmed. A preliminary mechanistic investigation indicates that the peptidomimetic works by a synergistic effect of conformational restriction and metal sequestration. Such peptidomimetics can shed light on the mechanism of aggregation and a novel therapeutic approach.